scholarly journals Crystal Structure of the ECH2 Catalytic Domain of CurF from Lyngbya majuscula

2007 ◽  
Vol 282 (49) ◽  
pp. 35954-35963 ◽  
Author(s):  
Todd W. Geders ◽  
Liangcai Gu ◽  
Jonathan C. Mowers ◽  
Haichuan Liu ◽  
William H. Gerwick ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Functional Group ◽  
Catalytic Domain ◽  
Acyl Carrier Protein ◽  
Cyclopropane Ring ◽  
Chemical Diversity ◽  
Site Directed Mutagenesis ◽  
Lyngbya Majuscula ◽  
Multifunctional Protein ◽  
Curacin A

Curacin A is a mixed polyketide/nonribosomal peptide possessing anti-mitotic and anti-proliferative activity. In the biosynthesis of curacin A, the N-terminal domain of the CurF multifunctional protein catalyzes decarboxylation of 3-methylglutaconyl-acyl carrier protein (ACP) to 3-methylcrotonyl-ACP, the postulated precursor of the cyclopropane ring of curacin A. This decarboxylase is encoded within an “HCS cassette” that is used by several other polyketide biosynthetic systems to generate chemical diversity by introduction of a β-branch functional group to the natural product. The crystal structure of the CurF N-terminal ECH2 domain establishes that the protein is a crotonase superfamily member. Ala78 and Gly118 form an oxyanion hole in the active site that includes only three polar side chains as potential catalytic residues. Site-directed mutagenesis and a biochemical assay established critical functions for His240 and Lys86, whereas Tyr82 was nonessential. A decarboxylation mechanism is proposed in which His240 serves to stabilize the substrate carboxylate and Lys86 donates a proton to C-4 of the acyl-ACP enolate intermediate to form the Δ2 unsaturated isopentenoyl-ACP product. The CurF ECH2 domain showed a 20-fold selectivity for ACP-over CoA-linked substrates. Specificity for ACP-linked substrates has not been reported for any other crotonase superfamily decarboxylase. Tyr73 may select against CoA-linked substrates by blocking a contact of Arg38 with the CoA adenosine 5′-phosphate.

scholarly journals Crystal structure of β-L-arabinobiosidase belonging to glycoside hydrolase family 121

2020 ◽  
Author(s):  
Keita Saito ◽  
Alexander Holm Viborg ◽  
Shiho Sakamoto ◽  
Takatoshi Arakawa ◽  
Chihaya Yamada ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Glycoside Hydrolase ◽  
Plant Pathogens ◽  
Catalytic Domain ◽  
Bifidobacterium Longum ◽  
Degradation Pathway ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Glycoside Hydrolase Family ◽  
Import System

AbstractEnzymes acting on α-L-arabinofuranosides have been extensively studied; however, the structures and functions of β-L-arabinofuranosidases are not fully understood. Three enzymes and an ABC transporter in a gene cluster of Bifidobacterium longum JCM 1217 constitute a degradation and import system of β-L-arabinooligosaccharides on plant hydroxyproline-rich glycoproteins. An extracellular β-L-arabinobiosidase (HypBA2) belonging to the glycoside hydrolase (GH) family 121 plays a key role in the degradation pathway by releasing β-1,2-linked arabinofuranose disaccharide (β-Ara2) for the specific sugar importer. Here, we present the crystal structure of the catalytic region of HypBA2 as the first three-dimensional structure of GH121 at 1.85 Å resolution. The HypBA2 structure consists of a central catalytic (α/α)6 barrel domain and two flanking (N- and C-terminal) β-sandwich domains. A pocket in the catalytic domain appears to be suitable for accommodating the β-Ara2 disaccharide; this pocket is highly conserved among GH121 proteins. The three acidic residues Glu383, Asp515, and Glu713, located in this pocket, are completely conserved among all ~270 members of GH121; site-directed mutagenesis analysis showed that they are essential for catalytic activity. The active site of HypBA2 was compared with those of GH63 α-glycosidase, GH94 chitobiose phosphorylase, GH142 β-L-arabinofuranosidase, GH78 α-L-rhamnosidase, and GH37 α,α-trehalase. Based on these analyses, we concluded that the three conserved residues are essential for catalysis and substrate binding. β-L-Arabinobiosidase genes in GH121 are mainly found in the genomes of bifidobacteria and Xanthomonas species, suggesting that the cleavage and specific import system for the β-Ara2 disaccharide on plant hydroxyproline-rich glycoproteins are shared in animal gut symbionts and plant pathogens.

scholarly journals Biochemical and Structural Insights Concerning Triclosan Resistance in a Novel YX7K Type Enoyl-Acyl Carrier Protein Reductase from Soil Metagenome

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Raees Khan ◽  
Amir Zeb ◽  
Kihyuck Choi ◽  
Gihwan Lee ◽  
Keun Woo Lee ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Fatty Acid Biosynthesis ◽  
Catalytic Domain ◽  
Acyl Carrier Protein ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Carrier Protein ◽  
The Novel ◽  
Triclosan Resistance ◽  
Structural Insights

Abstract Enoyl-acyl carrier protein reductase (ENR) catalyzes the last reduction step in the bacterial type II fatty acid biosynthesis cycle. ENRs include FabI, FabL, FabL2, FabK, and FabV. Previously, we reported a unique triclosan (TCL) resistant ENR homolog that was predominant in obligate intracellular pathogenic bacteria and Apicomplexa. Herein, we report the biochemical and structural basis of TCL resistance in this novel ENR. The purified protein revealed NADH-dependent ENR activity and shared similarity to prototypic FabI. Thus, this metagenome-derived ENR was designated FabI2. Unlike other prototypic bacterial ENRs with the YX6K type catalytic domain, FabI2 possessed a unique YX7K type catalytic domain. Computational modeling followed by site-directed mutagenesis revealed that mild resistance (20 µg/ml of minimum inhibitory concentration) of FabI2 to TCL was confined to the relatively less bulky side chain of A128. Substitution of A128 in FabI2 with bulky valine (V128) elevated TCL resistance. Phylogenetic analysis further suggested that the novel FabI2 and prototypical FabI evolved from a common short-chain dehydrogenase reductase family. To our best knowledge, FabI2 is the only known ENR shared by intracellular pathogenic prokaryotes, intracellular pathogenic lower eukaryotes, and a few higher eukaryotes. This suggests that the ENRs of prokaryotes and eukaryotes diverged from a common ancestral ENR of FabI2.

Characterization of Recombinant Human Coagulation Factor XFriuli

1996 ◽  
Vol 75 (02) ◽  
pp. 313-317 ◽  
Author(s):  
D J Kim ◽  
A Girolami ◽  
H L James
Keyword(s):  
Catalytic Domain ◽  
Coagulation Factor ◽  
Molecular Size ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Bleeding Tendency ◽  
Factor X ◽  
Amino Terminal ◽  
Normal Plasma ◽  
Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

scholarly journals Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1393
Author(s):  
Thanyaporn Dechtawewat ◽  
Sittiruk Roytrakul ◽  
Yodying Yingchutrakul ◽  
Sawanya Charoenlappanit ◽  
Bunpote Siridechadilok ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
Denv Infection ◽  
Site Directed Mutagenesis ◽  
Phosphorylation Sites ◽  
Post Translational Modification ◽  
Multifunctional Protein ◽  
Nonstructural Protein 1 ◽  
Underlying Mechanisms

Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design.

The first crystal structure of the peptidase domain of the U32 peptidase family

2015 ◽  
Vol 71 (12) ◽  
pp. 2505-2512 ◽  
Author(s):  
Magdalena Schacherl ◽  
Angelika A. M. Montada ◽  
Elena Brunstein ◽  
Ulrich Baumann
Keyword(s):  
Crystal Structure ◽  
Catalytic Mechanism ◽  
Quaternary Structure ◽  
Catalytic Domain ◽  
Three Dimensional ◽  
Zinc Ion ◽  
Crystal Structure Analysis ◽  
Dimensional Structure ◽  
Sequence Motifs ◽  
Conserved Sequence

The U32 family is a collection of over 2500 annotated peptidases in the MEROPS database with unknown catalytic mechanism. They mainly occur in bacteria and archaea, but a few representatives have also been identified in eukarya. Many of the U32 members have been linked to pathogenicity, such as proteins fromHelicobacterandSalmonella. The first crystal structure analysis of a U32 catalytic domain fromMethanopyrus kandleri(genemk0906) reveals a modified (βα)8TIM-barrel fold with some unique features. The connecting segment between strands β7 and β8 is extended and helix α7 is located on top of the C-terminal end of the barrel body. The protein exhibits a dimeric quaternary structure in which a zinc ion is symmetrically bound by histidine and cysteine side chains from both monomers. These residues reside in conserved sequence motifs. No typical proteolytic motifs are discernible in the three-dimensional structure, and biochemical assays failed to demonstrate proteolytic activity. A tunnel in which an acetate ion is bound is located in the C-terminal part of the β-barrel. Two hydrophobic grooves lead to a tunnel at the C-terminal end of the barrel in which an acetate ion is bound. One of the grooves binds to aStrep-Tag II of another dimer in the crystal lattice. Thus, these grooves may be binding sites for hydrophobic peptides or other ligands.

scholarly journals Crystal structure of an active form of RAS protein, a complex of a GTP analog and the HRAS p21 catalytic domain.

1990 ◽  
Vol 87 (12) ◽  
pp. 4849-4853 ◽  
Author(s):  
A. T. Brunger ◽  
M. V. Milburn ◽  
L. Tong ◽  
A. M. deVos ◽  
J. Jancarik ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Domain ◽  
Active Form ◽  
Protein A ◽  
Ras Protein

Crystal structure of the de-ubiquitinating enzyme UCH37 (human UCH-L5) catalytic domain

2009 ◽  
Vol 390 (3) ◽  
pp. 855-860 ◽  
Author(s):  
Kazuya Nishio ◽  
Sang-Woo Kim ◽  
Kentaro Kawai ◽  
Tsunehiro Mizushima ◽  
Takashi Yamane ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Domain

scholarly journals High resolution crystal structure of the catalytic domain of MCR-1

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Guixing Ma ◽  
Yifan Zhu ◽  
Zhicheng Yu ◽  
Ashfaq Ahmad ◽  
Hongmin Zhang
Keyword(s):  
Crystal Structure ◽  
High Resolution ◽  
Catalytic Domain
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